T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. In the inactivated state, these cells circulate in the blood and accumulate in lymphoid tissues such as lymph nodes and spleen. Upon encounter with antigens on specialized presenting cells (dendritic cells), these resting T-cells become activated and differentiate into effector cells. The effector cells leave the lymphoid tissues and blood, entering sites of infection to combat pathogens. They can also cause autoimmune pathology. After elimination of an infecting organism, most activated T-cells die, but some remain as memory cells. Some memory T-cells recirculate in lymphoid compartments and others patrol peripheral tissues. Other lymphocytes such as regulatory T cells contribute to suppression of these T-cell responses. This project attempts to gain both a qualitative (especially tissue-specific 4 dimensional space and time) and a quantitative understanding of the activation, differentiation, migration, cell-cell interaction, memory status, and reactivation properties of both CD4 and CD8 T-cells. The movement of activated T-cells into non-lymphoid tissues is being analyzed using imaging techniques that allow high-resolution dynamic observation of how cells migrate, interact, and carry out their effector functions, while a finer grained analysis of tissue architecture, T cell localization, and functional state are being assessed using a newly developed method for quantitative multiplex static imaging called Histo-cytometry. Through this research, a better understanding of lymphocyte dynamics during an immune response to infection or after vaccination or during an autoimmune response will be established. These new insights can contribute to the more effective design of vaccines and to strategies for the amelioration of autoimmune processes. We have established a robust system for vaccination using the non-replicating pox vector MVA, to permit in situ analysis of immune cell behavior in response to a clinically used vaccine vector. Our studies indicate unexpected locations the cells initially infected by MVA within draining lymph nodes. We find distinct dendritic cells involved in initial antigen presentation to CD4 vs. CD8 T-cells. Following initial activation of these T cell subsets in separate places within the lymph node on these distinct antigen presenting cells, both CD4 and CD8 T cells change their location and interact with the same dendritic cell type, the XCR1+ (CD8alpha+) cell; this communication is essential for robust memory CD8 T cell responses. These findings have important implications for how strong and persistent cell-mediated immune responses are generated, with relevance to vaccines against some viruses and against cancer. This project is also related to studies described in ZIA AI000545-28 LSB Multiscale Analysis of Immune Responses. In collaborative studies using our advanced method of 2-photon dynamic imaging and multiplex tissue analysis called Histo-cytometry, we showed with Dr. R. Caspi of the NEI that a circuit involving NK cells, T cells, and dendritic cells controlled the pathogenic activity of effector CD4 T cells. Our imaging studies helped develop an understanding of how these cells transmitted signals in situ through control of cell-cell interactions. With Dr. T. Veres, a former fellow, we have developed a novel method for imaging the trachea of mice. The trachea more closely resembles human small airways involved in asthma than do mouse small airways. Using this model, a combination of classical cell immunological analysis, dynamic intravital imaging and histochemistry has shown the clustering of small numbers of antigen activated T cells with dendritic cells and the local production of cytokines by only a small fraction of these cells at any one time, extending our previous reports showing the episodic effector function of antigen engaged T cells in tissues. We have also visualized local recruitment of eosinophils and local cytokine secretion based on pSTAT signatures. These data provide a new level of insight into the processes occurring over time to generate airway pathology in asthmatic individuals. In a project related to studies described in ZIA AI000545-28 LSB Multiscale Analysis of Immune Responses, we have used Histo-cytometry to discover that Treg cluster around mostly migratory dendritic cells together with Tconv cells. The latter in some cases are activated to produce IL-2 locally that induces pSTAT5 formation and signaling in the co-localized Tregs. The Treg clustering is TCR-dependent and selective deletion of TCRalpha in mature Tregs results on loss of clustering and rapid autoimmune disease development. Imaging shows that the Tregs in these clusters are the highest in suppressive molecules such as CD73 and CTLA-4 and also that IL-2 sensing contributes to the suppressive action of these cells. Strikingly, the same pSTAT5+ Treg clusters are seen at comparable numbers in germ-free and conventional SPF mice, indicating that the activation of Tconv to the IL-2 producing state involves self-recognition, emphasizing that thymic negative election does not eliminate all T cells with TCR able to respond overtly to self-ligands. These studies, performed in collaboration with A. Rudensky, thus reveal the architectural basis for homeostatic control of T cell autoreactivity and the role of both Treg TCR signaling and a negative feedback loop involving Tconv production of IL-2 that together maintain the host in a non-diseased state.